Abstract Studies of one of the most important tumor suppressor genes, RB1, have transformed the treatment of human cancer. This success was based on targeting E2F-responsive genes that were deregulated as a direct consequence of dysfunctional pRB. However, there is accumulating evidence of a multifaceted role of pRB and its involvement as a major regulator in processes outside the cell cycle. In particular, we rediscovered one of the first pRB-interacting proteins, KDM5A, in a screen for cellular factors critical for pRB function during differentiation. We found that KDM5A is directly downstream of pRB in the pathway, as decreasing KDM5A protein levels in RB-deficient cells was reminiscent of the effects of reintroducing pRB: it increased the activity of cell-type-specific transcription factors, and restored gene expression and morphological changes associated with differentiation. KDM5A is a demethylase that reads and removes methylation from lysine 4 on histone H3 (H3K4) at a subset of pRB target genes. Surprisingly, we found that pRB function at the onset of differentiation converges on activation of KDM5A target genes encoding mitochondrial components. Accordingly, overexpression in Rb-deficient cells of a major regulator of mitochondrial functions, PGC-1?, restored differentiation, phenocopying the reintroduction of pRB. In order to mechanistically understand the link between mitochondrial metabolic regulation and the induction of differentiation, we propose to study the RB pathway downstream of KDM5A: (1) we will investigate which functions in the mitochondrion are necessary and sufficient for differentiation rescue; (2) we will explore a therapeutic strategy for activation of mitochondrial function in cells from RB-deficient small cell lung cancers; (3) we will investigate whether there is a correlation between pRB deficiency and the mitochondrial signature, and how this relates to patient prognosis; and (4) we will study how mitochondrial genes are regulated by pRB, KDM5A and associated transcription factors. The relevance of KDM5A inhibition to cancer cell metabolism and differentiation is of great interest, as KDM5A is likely to be targeted by small-molecule inhibitors.